In the case of phospho and total 2plex, the capture/bead is specific, while the detection is common. Therefore, the phospho capture/bead should exclusively capture the phosphorylated protein, while the total capture/bead will capture all remaining non-phosphorylated proteins.
In theory, an increase in the phospho signal would likely cause a slight drop in the total protein signal. However, for every phospho/total 2-plex, a comparison between singleplex and multiplex is always conducted to demonstrate that both phospho and total signals are similar, regardless of whether they are run as singleplex or multiplex.






